The present invention relates to polymer gel patterning elements such as contact masks and diffusion masks and molding methods for making these patterning elements including complementary molding techniques.
In the fields of drug development, cellular biology, tissue engineering and physiology, there is now a need for devices with features on the size scale of individual cells. In particular, devices for controlling the spatial arrangement of assay materials in a format of arrays are important in these fields. In drug development, arrays are used to increase throughput and decrease the labor involved in screening compounds. High throughput screening requires parallel handling of chemical compounds, biochemical reagents, biological targets and other reagents, collectively referred to herein as biological materials.
Advances in high throughput screening depend upon automation, miniaturization and improved detection technology. Examples of biological assays that have been automated include ligand receptor binding assays, scintillation proximity assays, enzyme assays, enzyme-linked immunosorbent assays (ELISA), reporter gene assays, cell proliferation and toxicity assays, endotoxin assays and agglutination assays. The present invention relates most closely to miniaturization.
Younan Xia and George M. Whitesides, xe2x80x9cSoft Lithographyxe2x80x9d Angew. Chem. Ins. Ed. 37, 550-575 (1998) describes methods for patterning a variety of materials on surfaces using pattern-transfer elements made of polydimethylsiloxane (xe2x80x9cPDMSxe2x80x9d). In one extension of soft lithography, a PDMS contact mask is used to shield a substrate in all areas except where there are holes through the contact mask. Material is then passed through the holes and onto the exposed portions of the substrate.
In at least one significant way, PDMS pattern transfer elements are better adapted to patterning of inorganic materials, such as electroluminescent compounds for optical displays, such as is described in Duffy, et. al Adv. Material 1999, 11, 546-552, than they are for patterning biological materials; because PDMS is cytophilic. Consequently, when patterning cells using a PDMS patterning element, a large proportion of the cells intended for deposition onto the substrate are diverted because they adhere to the patterning element.
With the development of high throughput screening assay chips for use in drug discovery and medical research there is now a need for patterning elements that are non-cytophilic to complement, and in some instances to replace, PDMS elements used in these applications. The present invention meets this need by providing patterning elements made of materials with low cytophilicity and good biocompatibility.
In addition, polymers that form gels with water have properties similar to networks of proteins and polysaccharides that make up much of the extracellular material of the human body. This fact has spurred an interest in using hydrogels in medical implants. These materials may be well suited for creating an in vitro environment for the study of cells and other biological materials.
The handling of minute quantities of chemicals and biological materials as required for high throughput screening of drug candidates is a challenge currently facing the pharmaceutical industry. For instance, the lack of suitable liquid handling devices has been is cited as a stumbling block to further screen miniaturization. Stylli H; xe2x80x9cAn Integrated Approach to High Throughput Screeningxe2x80x9d in Handbook for the 1994 International Forum on Advances in Screening Technologies and Data Management at p. 5. Methods have been developed to position such minute quantities in a small area using robotic equipment and ink jet delivery devices. Commonly-assigned co-pending patent application Ser. No. 09/709,776 discloses alternative methods of positioning minute quantities of material in a small area on a biological array that are compatible with and complementary to robotic methods. One such method involves placing a contact mask over a substrate to conceal a portion of the substrate and leave a plurality of discontinuous portions of the substrate exposed. Such a mask has a plurality of holes through it. Each of the holes, together with the portion of the substrate surface which it overlies, forms a cavity. Biological and chemical materials can be deposited into each of the cavities individually using robotic equipment, or collectively by immersion in a solution, spraying, brushing or dropwise deposition using far less sophisticated and expensive equipment than is conventionally used to address individual elements of an array. PDMS has desirable adhesion, elasticity and strength properties and can be cast from a non-viscous precursor so that minute features like 50 xcexcm holes are transferred from a mold master to the PDMS. PDMS, however, is cytophilic, which is problematic for patterning cells, proteins and other biological materials in an array because these materials tend to adsorb onto the PDMS. In many applications for which bioarrays are suited, it is necessary to deposit material over the entire array or into a large group of adjacent cavities. Depositing material over a large contiguous portion of an array has potential cost savings in fabricating and using the array because these steps can be conducted without expensive and time consuming robotic manipulations. However, when large areas of the array are addressed collectively, it is detrimental to the process if the material adheres to the top surface of the contact mask instead of depositing into a cavity. In addition, material adsorbed by the PDMS in one patterning step may interfere or react with material being patterned in a subsequent step. The present invention provides a solution to this problem by providing a non-cytophilic patterning element, such as a contact mask, that has the desired properties of adhesion and elasticity (akin to those of PDMS) and acceptable tensile strength for use in patterning of biological and chemical materials on microarrays.
In accordance with one embodiment of the present invention, a polymer gel patterning element for patterning biological materials is provided. In another embodiment, the present invention provides a polymer gel contact mask. Yet another embodiment of the present invention provides a contact mask comprising a polymer gel having at least one hole therethrough. Another embodiment of the present invention comprises a polymer gel contact mask formed by complementary molding.
The present invention also provides a mold for producing a hydrogel contact mask from a precursor composition, the mold comprising first and second half molds wherein the first half mold is made from an elastomer and deforms to accommodate dimensional changes in the precursor composition as it cures into a hydrogel. In another embodiment, the present invention includes a mold comprising a half mold produced by thermal imprinting with an elastomeric master. The present invention further provides a complementary mold comprising first and second half molds each having molding surfaces, wherein the molding surfaces define a void when the first and second half molds are closed and wherein at least one of the first or second half molds is an elastomer. Yet another embodiment of the present invention includes a complementary mold comprising a PDMS half mold having a first molding surface, a release film adjacent to the first molding surface, and a rigid half mold having a second molding surface, wherein the first and second molding surfaces define a void.
The present invention further provides a method of complementary molding comprising the steps of providing a complementary mold including first and second half molds each having molding surfaces, wherein the molding surfaces define a void when the first and second half molds are closed and wherein at least one of the first or second half molds is an elastomer. Further wherein a polymerizable precursor is introduced, the half molds are closed, and the molding precursor is polymerized. Alternatively, the polymerizable precursor can be introduced into the void after the mold surfaces form the seal.
In another embodiment of the present invention, a method of complementary molding comprises the steps of providing a complementary mold including first and second half molds each having a molding surface, the molding surfaces defining a void there between, wherein the half molds close to form a void, and wherein at least one of the first or second components is an elastomer. The method further includes closing the half molds to form a void, introducing a polymerizable precursor into the void after the mold surfaces form the void, and polymerizing the molding precursor.
In yet another embodiment of the present invention, a method of continuous complementary molding comprises the steps of providing a first half mold including a plurality of first molding surfaces adapted and arranged for moving the molding surfaces from an unprocessed position to a processed position, providing a second half mold having a second molding surface, wherein the first and second molding surfaces form a void there between when closed, applying polymerizable polymer precursor to one of the molding surfaces in the unprocessed position, closing the half molds, polymerizing the polymer precursor, and moving the molded polymer to the processed position.
Another embodiment of the present invention includes a method of thermal imprinting comprising the steps of providing an elastomeric master having a first imprinting surface and a elastomeric substrate having a second imprinting surface, introducing a film having a lower glass transition temperature than the elastomeric master and the elastomeric substrate, between the elastomeric master and the elastomeric substrate, heating the film to a temperature above the glass transition temperature of the film, and cooling the film. Yet another embodiment of the present invention includes a method of forming a rigid complementary half mold comprising the steps of providing a first PDMS master having a first imprinting surface with surface features, providing a second PDMS substrate having a second imprinting surface, providing a polyethylene film between the first and second imprinting surfaces, applying pressure to the layered structure, heating the layered structure above the glass transition temperature of the polyethylene, and cooling the layered structure.